This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include dispensing droplets to a substrate from dispensers such as pin or capillaries (such as described in U.S. Pat. No. 5,807,522) or such as pulse jets (such as a piezoelectric inkjet head, as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere). For in situ fabrication methods, multiple different reagent droplets are deposited from drop dispensers at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array substrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivativesxe2x80x9d, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere
In array fabrication, the quantities of polynucleotide available, whether by deposition of previously obtained polynucleotides or by in situ synthesis, are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired target pattern, are of the correct size, and that the DNA is uniformly coated within the feature. Failure to meet such quality requirements can have serious consequences to diagnostic, screening, gene expression analysis or other purposes for which the array is being used.
However, in order to make arrays at a reasonable cost per array, it is also important that large numbers of arrays be fabricated in a short time. When drops are dispensed to form the arrays, this typically involves dispensing drops from a number of dispensers in co-ordination with scanning the dispensers in some pattern over a substrate (with one or more dispenser re-loadings, as desired). For example, drops for a portion of each array can be dispensed, the dispensers relocated, drops for the same portion of another array dispensed, and the process repeated followed by re-loading of the dispensers and repeating the foregoing sequence for another portion of all the arrays. However, such a pattern requires a large number of movements and hence a relatively long time to complete.
It would be desirable then, to provide a means for fabricating multiple arrays on a substrate while keeping the movement pattern of the multiple dispensers relatively simple.
The present invention provides in one aspect, a method of fabricating multiple arrays arranged successively in a first direction on a substrate. Each such array has multiple feature sets within the array which are also arranged successively in the first direction. The method uses a head system is used which has multiple successive sets of dispensers (for example, pulse jets such as piezoelectric or thermoelectric jets). In the method, the head system is advanced in the first direction over the substrate. Drop sets are dispensed from successive dispenser sets for each array in co-ordination with such movement, such that each drop set for multiple arrays are dispensed for each array.
In one aspect of the method, drop sets are dispensed from dispenser sets in an order the reverse of that from which the dispenser sets pass over a given location on the substrate as the head system advances in the first direction. In this aspect, each dispenser set deposits a drop set at a distance ahead of (as measured in the first direction) a drop set deposited by a preceding dispenser set which is less than the distance to the successive drop dispenser set which deposits the next drop set. Thus, while a given dispenser set is depositing drops for one feature set of an array, the dispenser sets which will deposit drops for successive feature sets of the same array have still not passed over the one feature set position of the same array (that is, they are still xe2x80x9cbehindxe2x80x9d the given dispenser set in relation to the direction of head advancement).
The arrays fabricated may have a distance between adjacent sets of features within the arrays, which is less than the distance between adjacent sets of dispensers. In fact, in one aspect of the present invention, arrays are fabricated by advancing and dispensing from the previously described head system, to obtain arrays with corresponding feature set spacing (for example, first feature set spacing) between adjacent arrays which is less than the total spacing (as measured in the direction of head advancement) of the dispenser sets which formed the arrays. Furthermore, the sets of features may extend in a direction transverse to the first direction. In this case, the method may additionally include moving the head in the transverse direction and dispensing the drop sets in co-ordination with such transverse movement so as to deposit drops along such feature sets.
The xe2x80x9cadvancingxe2x80x9d need not be a continuous motion, and in a particular aspect, the advancing and dispensing step may involve a number of sub-steps. Such sub-steps may include: while the head system is in one position in the first direction, depositing drop sets from at least one, and preferably multiple different dispenser sets for feature sets of different positions within multiple arrays; advancing the head system in the first direction to a next position; and repeating the foregoing two steps for successive feature sets within the arrays. During the repetitions, for each of the successive feature sets within the arrays, a corresponding dispenser set is used which deposited at a same feature set position of a previous array in step the first step during a previous cycle. This procedure may particularly be used in the case where the feature sets extend in a direction transverse to the first direction. In this case the method can additionally include moving the head in the transverse direction during the first step and dispensing the drop sets in co-ordination with such transverse movement so as to deposit drops along such feature sets.
Any desired number of successively arranged arrays can be fabricated on the same substrate by the method, each array having any desired number of features sets (for example, at least three, at least five, at least ten arrays, or at least twenty arrays each with at least three, at least five, at least ten, at least twenty, or at least one hundred feature sets).
The present invention can be applied to fabricating arrays of any chemical moieties, with any number of features within each set of an array being the same or different. The invention may, for example, be applied to fabricating arrays of monomeric moieties or polymers, such as biopolymers. (for example, polynucleotides or peptides). In the case of polymeric moieties, the drop sets may contain the polymeric moieties themselves (such as solutions of the polynucleotides or peptides) or monomeric moieties (such as nucleotides or amino acids) which may be reacted in sequence (through deposition of multiple different monomer containing moieties at each of the feature locations) to form the desired polymeric moieties. Any of the drops within a dispensed set of drops may be of the same or different composition, and any of the features within a feature set of the array may be the same or different composition (with any number of the feature sets being of the same or different composition).
Each feature set of the arrays may have multiple features arranged successively in the first direction. In this case, the dispenser sets may also be arranged successively in the first direction, with each set having multiple dispensers arranged successively within the set in a direction transverse to the first direction. For example, the feature sets may be rows of features arranged successively in the first direction (which implies, in the case of an array, that the individual rows extend lengthwise in a direction transverse to the first direction). The dispenser sets may also be arranged successively in the first direction with each set having multiple dispensers arranged successively within the set in a second direction transverse to the first. For example, the dispenser sets are rows of dispensers, each of which extends lengthwise in the transverse direction.
The present invention further provides an apparatus of the type described above in connection with any of the methods of the present invention. Such an apparatus may include a head system of a type already described above, and a transport system to advance the head system in the first direction with respect to a substrate. A processor communicates with the head system and transport system. The processor can advance the head system in the first direction over the substrate while coordinating drop dispensing from the head system with the advancement. In particular, the processor may dispense drop sets from successive dispenser sets for each array, such that each drop set for multiple arrays is deposited by a corresponding dispenser set which earlier deposited at a same feature set position within a previous array, in a manner as already described. The apparatus may optionally additionally include a loading station with receptacles to retain multiple different fluids such that the dispensers can be simultaneously brought into contact with respective receptacles for loading the dispensers with the different fluids. Each dispenser in such an apparatus may be constructed so that it holds no more than 100 xcexcl of a fluid to be dispensed as drops (or no more than 10 xcexcl or no more than 1 xcexcl of such fluid). By the dispenser holding a certain volume is referenced the entire dispenser including any reservoir in continuous communication with the remainder of the dispenser.
The present invention further provides a computer program for use with an apparatus such as already described above, having a head system, transport system, and processor. The computer program, when loaded into the processor, performs the steps of a method of the present invention (by controlling the apparatus, particularly the head system and transport system, appropriately). The computer program may be communicated to the processor (for example, from a remote location), or may be stored on a computer program product comprising a computer readable storage medium.
The present invention can allow for the fabrication of multiple arrays on a substrate in a relatively efficient manner with a simple movement pattern for the dispensers.